Lipoxin analogs and method for the treatment of periodontal disease

ABSTRACT

This invention provides new lipoxin analogs, compositions containing these analogs, and methods of using these compounds and compositions for treating and preventing oral inflammation, including gingivitis, periodontitis, and other forms of periodontal disease. The invention also provides for methods of treating and preventing oral inflammation, including gingivitis, periodontitis, and other forms of periodontal disease with compositions containing COX-2 inhibitors. Further, the invention provides methods for preventing systemic diseases beyond theoral cavity that are related to periodontal disease using the compositions containing lipoxin analogs, COX-2 inhibitors, or both.

RELATED APPLICATIONS

This application is a U.S. national phase application under 35 U.S.C.§371 of international application No. PCT/US01/09096 filed Mar. 20,2001, which international application claims priority to U.S.Provisional Patent Application No. 60/190,656, filed Mar. 20, 2000.International application No. PCT/US01/09096 was filed in English anddesignated the United States.

GOVERNMENT INTEREST

This invention was made with Government Support under Contract NumberDE13499 awarded by the National Institute of Dental & CraniofacialResearch. The Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to lipoxin compounds and COX-2 inhibitors.The present invention also relates to the treatment and prevention oforal inflammation, including gingivitis, periodontitis, and other formsof periodontal disease. Further, the invention relates to methods ofpreventing systemic diseases beyond the oral cavity that are related toperiodontal disease.

BACKGROUND OF THE INVENTION

Periodontal diseases, ranging from gingivitis to more severe forms ofperiodontitis, are initiated by a bacterial infection followed by a hostresponse that may lead to a highly degenerative oral disease includingtooth loss and tissue damage (Page, R. C. (1998) Ann. Periodontol. 3,108). The current treatments of periodontal diseases, which affect alarge percentage of the population, involve primarily the use ofcompositions containing antimicrobial compounds or various non-steroidalantiinflammatory agents (NSAIDs).

Although bacteria appear to be essential for the causation ofperiodontitis, progression of periodontal disease is dependent on thehost response to pathogens that colonize the tooth surface (Hart, T. C.,et al. (1994) J. Periodontol. 65, 521). In turn, periodontal disease canbe controlled chemotherapeutically by uncoupling host-mediateddestruction rather than reducing the etiological load (Offenbacher, S.et al. (1993) J. Periodontol. 64, 432). Along these lines, a body ofevidence has identified the inhibition of PGE₂ formation and itspresence at gingival sites as being relevant therapeutic interventions.For example, PGE₂ generation from gingival homogenates is significantlyinhibited by flurbiprofen (ElAttar, T. M. A., et al. (1984) J.Periodontol. 55, 536), and COX-derived eicosanoids in crevicular fluid(CF) are decreased in animals taking flurbiprofen (Smith, M. A., et al.(1993) Infection and Immunity 61, 1453; Offenbacher, S., et al. (1989)J. Periodontal Res. 24, 63). Flurbiprofen also reduced CF-PGE₂ levels,gingival inflammation, tooth attachment loss and bone loss, and in somecases resulted in bone gain (Pauletto, N. et al. (1997) J. Can. Dent.Assoc. 63, 824). In humans, flurbiprofen dramatically decreased theCF-PGE₂ levels (Abramson, M. M. et al. (1992) J. Periodont. Res. 27,539). These findings suggest that NSAIDs may exert their pharmacologicalaction of inhibiting COX derived proinflammatory eicosanoids within theperiodontium and suggest that novel anti-inflammatory agents might beuseful in managing periodontal diseases.

Polymorphonuclear leukocytes (PMN, neutrophils) are the most abundantimmune cells recruited to early inflammatory periodontal lesions and arethe most numerous host cells within the periodontal tissues (Hart, T.C., et al. (1994) J. Periodontol. 65, 521). The presence ofGram-negative oral pathogens represents the primary etiologic factor,however, the progression of periodontal disease is dependent on the hostresponse to pathogenic bacteria that colonize the tooth surface. Hence,recruitment of PMN followed by aberrant release of inflammatorymediators not only contributes to the onset of periodontal disease andis associated with rapid and widespread tissue destruction (Daniel, M.A., et al. (1996) J. Periodontol. 67,1070), but can also be furtheramplified by the release of an array of inflammatory mediators byneutrophils within the periodontium.

It is well known that PMN participate in host defense against bacterialinfections and are also involved in noxious inflammatory reactions(Weiss, S. J., et al. (1981) J. Clin. Invest. 68, 714; Babior, B. M.(1984) Blood 64, 959). Recruitment of neutrophils to the periodontiumcontributes to the progression of periodontal disease and to thedestruction of periodontal tissues (Page, R. C. (1998) Ann. Periodontol.3, 108; Daniel, M. A., et al. (1996) J. Periodontol. 67, 1070).

Several inflammatory mediators such as cytokines, chemokines andmetalloproteases are associated with periodontal disease (Romanelli, R.,et al. (1999) Infect. Immun. 67, 2319; Gainet, J., et al. (1998) Lab.Invest. 78, 755; Assuma, R., et al. (1998) J. Immunol. 160, 403). Otherprominent mediators are the arachidonic acid derived products, includingleukotriene B₄ (LTB₄) and prostaglandin E₂ (PGE₂) (Offenbacher, S. etal. (1986) J. Periodontal Res. 21, 101). Indeed, many of thepathophysiological events that occur in periodontal diseases can beexplained to a large extent by the activities of lipid mediators(Solomon, L. M., et al. (1968) J. Invest. Dermatol. 51, 280; Raisz, L.G., et al. (1974) Prostaglandins 8, 377; Klein, D. C., et al. (1970)Endocrinology 86, 1436; Crunkhorn, P., et al. (1969) Br. J. Pharmacol.36, 216; Collier, J. G., et al. (1972) Br. J. Pharmacol. 44, 374). Forexample, LTB₄, a well appreciated and potent chemoattractant, alsoinitiates the accumulation of leukocytes within inflamed sites,stimulates the release of granule-associated enzymes (Borgeat, P., etal. (1990) Clin. Biochem. 23, 459) and was recently found to stimulatebone resorption (Traianedes, K., et al. (1998) Endocrinology 139, 3178).

Along these lines, PGE2 is a very potent stimulator of bone loss, whichis held to be a hallmark of periodontal disease (Zubery, Y., et al.(1998) Infect. Immun. 66, 4158). PGE₂ is also well appreciated for itsability to directly mediate vasodilation, increase vascularpermeability, enhance pain perception by bradykinin and histamine, alterconnective tissue metabolism, and enhance osteodastic bone resorption(Tsai, C. -C. et al. (1998) J. Dentistry 26, 97). The levels of PGE₂ aresignificantly elevated in the crevicular fluid (CF) of patients withperiodontal infections, especially localized juvenile periodontitis,when compared to healthy sites. These levels correlate with diseaseseverity and aggressiveness and constitute a reliable indicator ofongoing clinical periodontal tissue destruction (Offenbacher, S., et al.(1984) J. Periodontal Res. 19, 1). CF-PGE₂ levels can also be used topredict future acute loss of periodontal attachment (Offenbacher, S., etal. (1986) J. Periodontal Res. 21, 101).

Pathophysiological responses that occur in periodontal diseases,including inflammatory cell recruitment, edema, pain, bone resorptionand collagen destruction, can be mediated for the most part by effectormolecules originating from the arachidonate cascade (Solomon, L. M. etal. (1968) J. Invest. Dermatol. 51,280; Raisz, L. G., et al. (1974)Prostaglandins 8,377; Klein, D. C., et al. (1970) Endocrinology 86,1436; Crunkhorn, P., et al. (1969) Br. J. Pharmacol. 36, 216; Collier,J. G., et al. (1972) Br. J. Pharmacol. 44, 374). In particular,considerable evidence has demonstrated the importance of PGE₂ in thepathogenesis of periodontal diseases. In vitro, PGE₂ increasesosteoclast numbers and bone resorption (Lader, C. S., et al. (1998)Endocrinology 139, 3157), decreases proteoglycan synthesis and increasesmetalloprotease production by cultured chondrocytes (Debrumfernandes, A.J., et al. (1996) Br. J. Pharmacol. 188, 1597). Bone resorption in vivocaused by three periodontal pathogens is mediated in part by PGE₂,causing tooth attachment loss and bone loss (Zubery, Y., et al. (1998)Infect. Immun. 66, 4158). Prior to these findings, PGE₂ was proposed asa reliable molecular indicator of ongoing periodontal tissue destructionthat might be used to predict future acute periodontal attachment loss(Offenbacher, S., et al. (1986) J. Periodontal Res. 21, 101).

Prostaglandin endoperoxide synthase (cyclooxygenase, COX) catalyzes tworeactions by which arachidonic acid is converted to PGH₂, the commonprecursor of all prostanoids including PGE₂. To date, two COX isoformsare known (Smith, W. L., et al. (1996) J. Biol. Chem. 271, 33157). COX-1appears to support the levels of prostanoid biosynthesis required formaintaining organ and tissue homeostasis (Smith, W. L., et al. (1996) J.Biol. Chem. 271, 33157; Vane, J. R., et al. (1996) Scand. J. Rheumatol.102, 9), whereas COX-2 expression appears to be restricted in basalconditions within most tissues and is up-regulated during inflammationor stress in a wide range of tissues (O'Banion, M. K., et al. (1992)Proc. Natl. Acad. Sci. USA 89, 4888; Seibert, K., et al. (1994) Proc.Natl. Acad. Sci. USA 91, 12013; Needleman, P., et al. (1997) J.Rheumatol. 24, 6). The finding that homogenates of inflamed periodontaltissues display an increased PGE₂ synthetic capacity when compared tohomogenates from healthy tissues suggests an increased COX activity isassociated with periodontal tissues (ElAttar, T. M. A. (1976)Prostaglandins 11, 331; Albers, H. K., et al. (1979) Dtsch. Zahnarztl.Z. 34, 440; ElAttar, T. M. A., et al. (1982) Prostaglandins Leukot. Med.8, 447; ElAttar, T. M. A., et al. (1984) J. Periodontol. 55, 536).Moreover, given the clearly deleterious actions of PGE₂ on the integrityof tissues of the periodontal pocket, both the potential involvement ofthe inducible COX isoform (COX-2) in periodontal disease and potentialrole of novel lipid mediators are of interest in the pathogenesis ofperiodontal disease.

Lipoxins (LX) and aspirin-triggered LX (ATL) are arachidonicacid-derived bioactive lipids that are formed by interactions betweenindividual lipoxygenases (LO) and appear to play an important role indownregulating neutrophil responses in inflammation (Serhan, C. N.(1997) Prostaglandins 53, 107). In the nanomolar range, LXA₄ and its 15Repimer (15-epi-LXA₄) triggered by-aspirin each inhibit fMLP- andLTB₄-stimulated PMN adhesion and transmigration and hence representpotential counterregulatory signals operative in the resolution ofinflammatory sites (Serhan, C. N. (1997) Prostaglandins 53,107; Serhan,C. N., et al. (1996) FASEB J. 10, 1147; Takano, T., et al. (1997) J.Exp. Med. 185, 1693; Serhan, C. N. et al. (1995) Biochemistry 34,14609). Like most autacoids and lipid mediators, LX are rapidlygenerated, act within a local microenvironment, and are rapidlyenzymatically inactivated. The roles of LX and ATL roles in vivo, werestudied by using metabolically stable LX and ATL analogs that weredesigned to resist rapid enzymatic inactivation and mimic the in vitroactions of naturally occurring LX and ATL (Serhan, C. N., et al. (1995)Biochemistry 34, 14609).

In addition to confirming the presence of LTB₄ and PGE₂, (Tsai, C. -C.et al. (1998) J. Dentistry 26, 97), it was shown for the first time thatLXA₄ is produced by activated neutrophils from LJP patients. It was alsoshown that LXA₄ is present within the crevicular fluid fromperiodontitis patients with active disease. These results are the firstdemonstration that LJP peripheral blood neutrophils are in a primedstate for LX generation. This in vivo “priming” for up-regulated lipoxinprofiles was also observed with neutrophils isolated from asthmaticpatients (Chavis, C., et al. (1996) J. Exp. Med. 183, 1633) and can bemimicked in vitro with cytokine-priming of neutrophils from healthydonors (Fiore, S., et al. (1990) J. Exp. Med. 172, 1451).

It was recently reported that LXA₄ and ATL analogs reduce leukocytetrafficking stimulated by TNF-α while concomitantly re-orientating thecytokine-chemokine aids towards an anti-inflammatory profile (Hachicha,M., et al. (1999) J. Exp. Med. 189,1923). LX-ATL can thus protect hosttissues via multilevel regulation of proinflammatory signals.

Periodontal disease has implications beyond the deleterious effects onoral tissues and structural integrity. Thus, periodontitis represents apotential risk factor for increased morbidity or mortality for severalsystemic conditions including cardiovascular diseases, pregnancycomplications, and diabetes (Page, R. C. (1998) Ann. Periodontol. 3,108; Garcia, R. I., et al. (1998) Ann. Periodontol. 3, 339). Of greatimportance in this context, is the finding that the systemic presence ofP. gingivalis up-regulates the expression of COX-2 (heart and lungs;FIG. 6) which is a marker of on-going inflammation (Herschman, H. R.(1998) Trends Cardiovasc. Med. 8, 145).

The recognition of the endogenous and multifaceted anti-inflammatoryrole of the lipoxins (Serhan, C. N. (1994) Biochim. Biophys. Acta, 1212,1; Serhan, C. N. (1997) Prostaglandins 53, 107), combined with thefindings that both lipoxin A4 and lipoxin B4 are rapidly deactivated bydehydrogenation (Serhan, C. N.; et al. (1993) Biochemistry, 32, 6313;Maddox, J. F. et al. (1998) FASEB J., 12, 487) or ω-oxidation (Sumimoto,H. et al. (1993) FEBS Lett., 315, 205; Mizukami, Y. et al. (1993)Biochim. Biophys. Acta, 1168, 87; Mizukami, Y. et al. (1994) Eur. J.Biochem, 224, 959), led to the design and synthesis of a number of LXanalogs with increased biostability (Serhan, C. N. et al. (1994)Biochemistry, 34, 14609). Several LX analogs of this type were reportedto have interesting biological properties and therapeutic potential(Serhan, C. N. et al. (1994) Biochemistry, 34, 14609; Takano, T. et al.(1998) J. Clin. Invest. 101, 819). The use of lipoxin analogs for thetreatment and prevention of periodontal disease as well as relatedsystemic diseases, however, has not been described previously.

SUMMARY OF THE INVENTION

The present invention provides new lipoxin analogs, compositionscontaining these analogs, and methods of using these compounds andcompositions for treating and preventing oral inflammation, includinggingivitis, periodontitis, and other forms of periodontal disease. Inone embodiment, the these new compounds are structural analogs ofbiostable lipoxin compounds, such as lipoxin A₄, lipoxin B₄, or otherrelated lipid mediators. Acceptable analogs include, but are not limitedto, structural analogs of two series of lipoxins: LXA series(LXA₄/15-epi-LXA₄) and LXB series (LXB₄/15-epi-LXB₄).

The lipoxin analogs of the present invention contain four majorcomponents: (a) the carboxyl component, (b) the diol component, (c) thetetraene component, and (d) the alcohol component. Each of thesecomponents can possess a number of structural variations and stillretain the key features necessary for lipoxin activity. Preferredcompounds of the present invention generally belong either to the LXAseries or the LXB series and can have structural modifications in one ormore of the above components.

The compositions containing the lipoxin analogs can be in any formsuitable for administration to a human or animal. Preferred forms of thecompositions are those that can be administered topically to the oralcavity, for example, solutions, suspensions, dispersions, ointments,creams, pastes, powders, such as tooth powders, toothpastes, gels,lozenges, salve, chewing gum, mouth sprays, pastilles, sachets,mouthwashes, aerosols, tablets, capsules, and floss conjugated with LXanalogs.

In another embodiment, the present invention provides methods fortreating and preventing oral inflammation, including gingivitis,periodontitis, and other forms of periodontal disease with compositionscontaining COX-2 inhibitors. For example, compositions containing suchcompounds as celecoxib, rofecoxib, and valdecoxib, can be administeredto a human or animal in manners similar to those for administration ofthe lipoxin analog containing compositions.

Further, the invention provides methods for preventing systemic diseasesbeyond the oral cavity that are related to periodontal disease using thecompositions containing lipoxin analogs, COX-2 inhibitors, or both. Suchdiseases include cardiovascular diseases, pregnancy complications, anddiabetes.

Therefore, it is an object of the present invention to provide lipoxinanalogs, such as LXA and LXB analogs having lipoxin activity.

It is also an object of the present invention to provide compositionscomprising lipoxin analogs, such as LXA and LXB analogs having lipoxinactivity.

It is a further object of the present invention to provide compositionscomprising lipoxin analogs and COX-2 inhibitors.

It is yet another object of the present invention to provide methods forthe treatment and prevention of oral inflammation, such as gingivitis,periodontitis, and other periodontal diseases.

It is another object of the present invention to provide methods for thetreatment and prevention of aphthous ulcers.

It is a further object of the present invention to provide methods forthe treatment and prevention of herpetic stomatitis.

It is an object of the present invention to provide methods for thetreatment and prevention of oral inflammation with compositionscomprising lipoxin analogs.

It is another object of the present invention to provide methods for thetreatment and prevention of oral inflammation with compositionscomprising COX-2 inhibitors.

It is yet another object of the present invention to provide methods forthe treatment and prevention of oral inflammation with compositionscomprising lipoxin analogs and COX-2 inhibitors.

It is an object of the present invention to provide methods for thetreatment of systemic diseases associated with periodontal disease.

It is a further object of the present invention to provide methods forthe treatment of cardiovascular diseases, pregnancy complications, anddiabetes.

It is another object of the present invention to provide methods for thetreatment of conditions associated with Porphyromonas gingivalis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that Activated PMN from LJP patients, but not fromasymptomatic controls, generated LXA₄.

FIG. 2 demonstrates that P. gingivalis elicits leukocyte infiltration invivo.

FIG. 3 illustrates that P. gingivalis induces COX-2 expression in humanPMN.

FIG. 4 shows that LXA₄ analogs inhibit leukocyte infiltration in vivo.

FIG. 5 illustrates that the aspirin-triggered LX-analog, 15R/S-methylLXA₄, inhibits P. gingivalis-induced PGE₂ production in murine airpouch.

FIG. 6 demonstrates that P. gingivalis causes systemic up-regulation ofCOX-2 mRNA.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Generally, the present invention provides new lipoxin analogs,compositions containing these analogs, and methods of using thesecompounds and compositions for treating and preventing oralinflammation, including gingivitis, periodontitis, aphthous ulcers,herpetic stomatitis, and other forms of periodontal disease. Theinvention also provides for methods of treating and preventing oralinflammation, including gingivitis, periodontitis, aphthous ulcers,herpetic stomatitis, and other forms of periodontal disease withcompositions containing COX-2 inhbitors. Further, the invention providesmethods for preventing systemic diseases beyond the oral cavity that arerelated to periodontal disease using the compositions containing lipoxinanalogs, COX-2 inhibitors, or both.

In one embodiment, the present invention provides new lipoxin compoundsthat are structural analogs of biostable lipoxin compounds, such aslipoxin A₄, lipoxin B₄, or other related lipid mediator. The termstructural analog as used herein means any molecule having the basicstructural components of lipoxin compounds. That is compounds containinga carboxyl component, a diol component, a tetraene component, and analcohol component. These components can be any size and can be joined toone another in any manner. Additionally, these components can containvarious substituents or have some of their carbon atoms replaced, forexample, by rings or heteroatoms. The compounds of the present inventionretain lipoxin activity. However, the compounds of the present inventiondo not undergo the typical metabolic deactivation of the parent lipoxincompounds. Thus, the in vivo half life of the compounds of the presentinvention is significiantly greater than the half life of the parentcompounds. Acceptable analogs include, but are not limited to,structural analogs of two series of lipoxins: LXA series (LXA4/15-epi-LXA4) and LXB series (LXB₄/15-epi-LXB4).

The lipoxin analogs of the present invention contain four majorcomponents: (a) the carboxyl component, (b) the diol component, (c) thetetraene component, and (d) the alcohol component. Each of thesecomponents can possess a number of structural variations and stillretain the key features necessary for lipoxin activity. Preferredcompounds of the present invention generally belong either to the LXAseries or the LXB series and can have structural modifications in one ormore of the above components. The following diagram provides the generalformulas for lipoxin compounds of the LXA and LXB series.

In a preferred embodiment of the present invention, the lipoxin analog aone of the following structures bearing the designated stereochemistry:

In these structures, the R-groups are independently selected as follows:

-   -   R is hydrogen or a straight, branched, cyclic, saturated, or        unsaturated alkyl;    -   R¹, R², R¹², R¹³ are each independently selected from        -   hydrogen;        -   straight, branched, cyclic, saturated, or unsaturated alkyl            having from 1 to 20 carbon atoms;        -   substituted alkyl having from 1 to 20 carbon atoms, wherein            the alkyl is substituted with one or more substituents            selected from halo, hydroxy, lower alkoxy, aryloxy, amino,            alkylamino, dialkylamino, acylamino, arylamino,            hydroxyamino, alkoxyamino, alkylthio, arylthio, carboxy,            carboxamido, carboalkoxy, aryl, and heteroaryl;        -   substituted aryl or heteroaryl wherein the aryl or            heteroaryl is substituted with one or more substituent            selected from alkyl, cycloalkyl, alkoxy, halo, aryl,            heteroaryl, carboxyl, and carboxamido; and        -   a group Z-Y,            -   wherein Z is a straight, branched, cyclic, saturated, or                unsaturated alkyl having from 1 to 20 carbon atoms;                substituted lower alkyl wherein the alkyl is substituted                with one or more substituents selected from halo,                hydroxy, lower alkoxy, aryloxy, amino, alkylamino,                dialkylamino, acylamino, arylamino, hydroxyamino,                alkoxyamino, alkylthio, arylthio, carboxy, carboxamido,                carboalkoxy, aryl, and heteroaryl; substituted aryl or                heteroaryl wherein the aryl or heteroaryl is substituted                with one or more substituents selected from alkyl,                cycloalkyl, alkoxy, halo, aryl, heteroaryl, carboxyl,                and carboxamido; and            -   Y is selected from hydrogen; alkyl; cycloalkyl;                carboxyl; carboxamido; aryl; heteroaryl; substituted                aryl or heteroaryl wherein the aryl or heteroaryl is                substituted with one or more substituents selected from                alkyl, cycloalkyl, alkoxy, halo, aryl, heteroaryl,                carboxyl, and carboxamido;    -   R³ is selected from hydrogen; straight, branched, cyclic,        saturated, or unsaturated alkyl having from 1 to 20 carbon        atoms;        -   substituted alkyl having from 1 to 20 carbon atoms, wherein            the alkyl is substituted with one or more substituents            selected from the group consisting of halo, hydroxy, lower            alkoxy, aryloxy, amino, alkylamino, dialkylamino, acylamino,            arylamino, hydroxyamino, alkoxyamino, alkylthio, arylthio,            carboxy, carboxamido, carboalkoxy, aryl, and heteroaryl;        -   substituted aryl or heteroaryl, wherein the aryl or            heteroaryl is substituted with one or more substituents            selected from the group consisting of alkyl, cycloalkyl,            alkoxy, halo, aryl, heteroaryl, carboxyl, and carboxamido;            and    -   R⁴-R¹¹ are selected from a group consisting of:        -   hydrogen;        -   halo;        -   straight, branched, cyclic, saturated, or unsaturated alkyl            having from 1 to 20 carbon atoms;        -   substituted alkyl haivng from 1 to 20 carbon atoms, wherein            the alkyl is substituted with one or more substituents            selected from halo, hydroxy, lower alkoxy, aryloxy, amino,            alkylamino, dialkylamino, acylamino, arylamino,            hydroxyamino, alkoxyamino, alkylthio, arylthio, carboxy,            carboxamido, carboalkoxy, aryl, and heteroaryl;        -   substituted aryl or heteroaryl wherein the aryl or            heteroaryl are substituted with one or more substituent            selected from alkyl, cycloalkyl, alkoxy, halo, aryl,            heteroaryl, carboxyl, and carboxamido;    -   R, R¹-R¹³ may be also connected to form one or more rings        containing 3 to 20 carbon atoms, 1 to 6 oxygen atoms or 1 to 6        nitrogen atoms.    -   A pair selected among the R¹-R¹³ groups may also be replaced        with a bond that generates a carbon-carbon double or triple bond        or a ring.

Examples of preferred compounds of the present invention are shown inScheme 1. These examples are provided for purposes of illustration andin no way limit the scope of the present invention. Also contemplated aspreferred compounds are the compounds shown in Scheme 1 wherein thecarbon chains and rings shown in the structures additionally possesssubstituents selected from halo, hydroxy, lower alkoxy, aryloxy, amino,alkylamino, dialkylamino, acylamino, arylamino, hydroxyamino,alkoxyamino, alkylthio, arylthio, carboxy, carboxamido, carboalkoxy,aryl, and heteroaryl.

Scheme 1 LXA₄ Series 15-epi-LXA₄ Series 1. Isomeric derivatives

2. Substituted tetraenes

R = Me, Ph 3. Ring-substituted tetraenes

4. Benzo-substituted derivatives

5. Derivatives substituted at the alcohol or diol components

Y = CH₂ or O

R = Me or CF₃ n = 1-10 6. Hydroxy-replacement derivatives

7. Carboxy-replacement derivatives

n = 1-10 R = polymeric material LXB₄ Series 15-epi-LXB₄ Series 1.Isomeric derivatives

2. Substituted tetraenes

R = Me, Ph 3. Ring-substituted tetraenes

4. Benzo-substituted derivatives

5. Derivatives substituted at the alcohol or diol components

Y = CH₂ or O

6. Hydroxy-replacement derivatives

7. Carboxy-replacement derivatives

The lipoxin analogs of the present invention can be manufactured usingconventional methods for producing the parent lipoxin compounds. Thereactions necessary to add various functional groups, rings, bonds, andheteroatoms to the present compounds are well within the skill of theordinary artisan.

In another embodiment, the present invention provides compositionscontaining these new lipoxin analogs. The new lipoxin analogs of thepresent invention can be formulated into compositions that are suitablefor administration to a human or animal. The compounds can beadministered by any suitable means. In general, suitable means ofadministration include, but are not limited to, topical, transdermal,oral, sublingual, nasal, buccal, rectal, and parenteral (e.g.,intravenous, subcutaneous or intramuscular) routes. In addition, thecompositions can be incorporated into or covalently attached to polymersfor topical use or for sustained delivery. The preferred method ofadministration is topical, for example, topical delivery to the oralcavity.

The compositions of the present invention can be in any form. Theseforms include, but are not limited to, solutions, suspensions,dispersions, ointments, creams, pastes, gels, powders, including toothpowders, toothpastes, lozenges, salve, chewing gum, mouth sprays,pastilles, sachets, mouthwashes, aerosols, tablets, capsules,transdermal patch, suppositories, and floss conjugated with LX analogs.Preferred forms of the compositions are those that can be administeredtopically to the oral cavity. Additionally, the lipoxin analogs of thepresent invention can be conjugated with or covalently linked topolymers, such as those that are conventionally used for the manufactureof dental floss. The lipoxin analogs can also be incorporated intopolymers or biopolymers for the sustained release of the compounds.Further, the lipoxin analogs can be incorporated into liposomes forsustained release delivery.

The compositions of the invention can include other components. Thesecomponents include, but are not limited to, pharmaceutical carriers,binders, fillers, flavorants, and stabilizers. Additionally, thecompositions of the present invention can contain additional activeingredients. For example, the compositions can contain COX-2 inhibitorsin addition to the lipoxin analogs. The present invention also providespharmaceutical compositions comprising COX-2 inhibitors in the absenceof the lipoxin analogs.

In yet another embodiment, the present invention provides methods forthe treatment and prevention oral inflammation, including gingivitis,periodontitis, aphthous ulcers, herpetic stomatitis, and other forms ofperiodontal disease with compositions containing the lipoxin analogs ofthe invention. These methods comprise administering to the patienthaving such a disease, a composition that comprises a lipoxin analog, aCOX-2 inhibitor, or both a lipoxin analog and a COX-2 inhibitor.

The present inventors have identified for the first time byLC/MS/MS-based analyses, that eicosanoids are generated by peripheralblood neutrophils from periodontitis (e.g. LJP) patients. The presentinventors have also found that LXA₄, PGE₂, and LTB₄ are present in humanCF. Experiments revealed that CF from localized juvenile periodontitis(LJP) patients contain prostaglandin (PG)E₂ and 5-lipoxygenase-derivedproduct, leukotriene B₄, and the biosynthesis interaction product,lipoxin (LX)A₄. Neutrophils from peripheral blood of LJP patients, butnot from asymptomatic donors, also generate LXA₄ suggesting a role forthis immunomodulatory molecule in periodontal disease.

Further, examination of an animal model of leukocyte trafficking andactivation of Porphyromonas gingivalis, an oral microbe clinicallyassociated with periodontal disease, showed that this microbe potentlyand rapidly (<4 hr) attracts large numbers of leukocytes, primarilyneutrophils (>80%) in vivo. These data indicate that this mediator,PGE₂, might originate in part from the infiltrating leukocytes.

P. gingivalis also up-regulates the expression of COX-2 frominfiltrating leukocytes. In addition, human PMN exposed to P. gingivalisalso stimulates the expression of COX-2 (FIG. 3). These results indicatethat the periodonal pathogens can attract leukocytes in vivo and induceleukocyte-COX-2. In light of the results described herein, the inducibleCOX isoform expressed in recruited leukocytes (local exudate) may be amajor source responsible for production of PGE₂ found in the CF ofperiodontal disease patients (FIGS. 1, 3, and 5).

Given the large number of PMN recruited at inflammatory lesions inperiodontal disease, the results described herein identify the PMN as apotential cellular primary target for therapeutic intervention.Metabolically stable lipoxin analogs (LX-ATL) have the potential ofblocking PMN infiltration as well as reducing COX-2 derived PGE₂ presentin gingival tissues. In this regard, topical application of the novellipoxin analogs of the present invention, or of anti-neutrophil agentsas described by Takano (Takano, T., et al. (1998) J. Clin. Invest. 101,819; Hachicha, M. et al. (1999) J. Exp. Med. 189, 1923) as well as newselective COX-2 inhibitors as described by (Needleman, P. et al. (1997)J. Rheumatol. 24, 6; Herschman, H. R. (1998) Trends Cardiovasc. Med. 8,145; Golden, B. D. et al. (1999) Rheum. Dis. Clin. North Am. 25, 359),may prove to be advantageous in this disease and associated pain sinceit could eliminate potential unwanted side-effects (particularly renaleffects in the elderly) associated with conventional methods of systemicdelivery, such as with nonsteroidal antiinfiammatory drugs.

The present inventors have determined that metabolically stable lipidmediators can be used to control host response in periodontal diseaseand related conditions. Studies confirmed that topical administration ofmetabolically stable analogs of LX or ATL within the pouch cavity ofmice potently blocked P. gingivalis induced neutrophil traffic in thedorsal air pouch model and lowered PGE₂ levels within exudates.

Together, the above findings identify PMN as an additional andpotentially important source of PGE₂ in periodontal tissues. Moreover,they provide evidence for a novel protective role for LX inperiodontitis, limiting further PMN recruitment and PMN-mediated tissueinjury that can lead to loss of inflammatory barriers that preventsystemic tissue invasion of oral microbial pathogens.

Therefore, the present invention provides methods for treating orpreventing oral inflammation, such as gingivitis, periodontitis,aphthous ulcers, herpetic stomatitis, and other periodontal diseases byadministering compositions containing the novel lipoxin compounds of thepresent invention. The active ingredient can be delivered over a widerange of doses depending upon the method of delivery and the conditionto be treated or, prevented. In general, active doses between about1×10⁻¹⁵ grams and 1×10⁻³ grams are useful in the present invention. Thecompositions can be any of those described above and are preferablyadministered via topical delivery in the oral cavity.

The present invention also provides a method for the treatment orprevention of oral inflammation, including gingivitis, periodontitis,aphthous ulcers, herpetic stomatitis, and other periodontal diseases byadministering compositions containing COX-2 inhibitors. Examples ofCOX-2 inhibitors include, but are not limited to, celecoxib, rofecoxib,and valdecoxib.

Advantageously, the present invention provides methods for treating orpreventing oral inflammation, including gingivitis, periodontitis,aphthous ulcers, herpetic stomafitis, and other periodontal diseases byadministering compositions containing both lipoxin analogs of thepresent invention and a COX-2 inhibitor.

In view of their inhibitory impact on neutrophil recruitment andsecondarily on PGE₂ levels, the lipoxin analogs of the present inventionare beneficial to the host not only in the context of periodontitis, butalso in a number of diseases which involve excessive PMN responses thatcan lead to losses in inflammatory barriers and increase invasion ofsystemic microbes. Blood borne P. gingivalis gives significant increasesin the murine tissue levels of COX-2 mRNA associated with both heart andlungs, supporting a potential role for this oral pathogen in theevolution of systemic events.

The concept that a local infection by P. gingivalis may have a systemicimpact on the status of the immune system, is further substantiated bythe finding that P. gingivalis injected in the air pouch up-regulatesCOX-2 mRNA levels in the lung-associated tissues. In view of theseresults, an effective treatment of periodontal conditions is likely tohave a beneficial impact on the prognosis of a number of systemicdiseases.

Thus, the present invention is also related to methods for treatingsystemic diseases that are related to periodontal disease, such ascardiovascular diseases, pregnancy complications, and diabetes. Thesemethods comprise administering to the patient having such a disease, acomposition that comprises a lipoxin analog, a COX-2 inhibitor, or botha lipoxin analog and a COX-2 inhibitor.

EXAMPLES

The present invention is further illustrated and supported by thefollowing examples. However, these examples should in no way beconsidered to be further limit the scope of the invention. To thecontrary, one having ordinary skill in the art would readily understandthat there are other embodiments, modifications, and equivalents of thepresent invention without departing from the spirit of the presentinvention and/or the scope of the appended claims.

Example 1 Identification of Eicosanoids From Human Poly-MorphonuclearLeukocytes in Juvenile Periodontitis

It was previously suggested that localized juvenile periodontitis (LJP)patients present altered lipid metabolism, including the 15-LO pathway(Noguchi, Z., et al. (1988) Prostaglandins Leukotrienes and EssentialFatty Acids 33, 137). Therefore, we evaluated the capacity of peripheralblood PMN from LJP patients and periodontal disease from age, sex, andrace matched controls to produce 5- and 15-LO-derived eicosanoids, aswell as the LO interaction product LXA₄.

Human polymorphonuclear leukocytes (PMN) from healthy volunteers andpatients with juvenile periodontitis were obtained by gradientcentrifugation of heparinized fresh venous blood using the method ofBöyum (Böyum, A. (1968) Scand. J. Clin. Lab. Invest. Suppl. 21, 77).Resulting granulocyte suspensions contained fewer than 0.2% monocytes asdetermined by esterase staining, and viability was greater than 96% asdetermined by trypan blue dye exclusion.

Freshly isolated PMN (5×10⁶ cells) were suspended in 0.5 ml Hank'sbuffered saline with 1.6 mM Ca²⁺ and incubated with A23187 (4 μM) at 37°C. for 20 minutes. The incubations were stopped with two volumes of coldmethanol and kept at −20° C. overnight. Protein precipitates werepelleted by centrifugation and washed twice with methanol. Thesupernatants were pooled, and the eicosanoids were extracted withExtract-Clean solid phase cartridges (500 mg C₁₈, Alltech AssociatesInc., Deerfield, Ill.), using PGB₂ ([M−H]⁻-=m/z 333) as an internalstandard for extraction recovery calculations (Takano, T., et al. (1998)J. Clin. Invest. 101, 819). The methyl formate fractions were taken todryness with a gentle stream of nitrogen and suspended in mobile phasefor LC/MS/MS analyses. LC/MS/MS was performed employing an LCQ (FinniganMAT, San Jose, Calif.) quadrupole ion trap mass spectrometer systemequipped with an electrospray ionization probe. Samples were injectedinto the HPLC component, comprised of a SpectraSYSTEM P4000 (ThermoSeparation Products, San Jose, Calif.) quaternary gradient pump, a LUNAC18-2 (150×2 mm, 5 μm) column, and a SpectraSYSTEM UV2000 (ThermoSeparation Products, San Jose, Calif.) UV/VIS absorbance detector. Thecolumn was eluted isocratically for 20 minutes withmethanol/water/acetic acid (65:34.99:0.01, v/v/v) at 0.2 ml/min,followed by a 20 minute linear gradient to methanol/acetic acid(99.99:0.01, v/v), and into the electrospray probe. The spray voltagewas set to 5-6 kV and the heated capillary to 250° C. Eicosanoids werequantitated by selected ion monitoring (SIM) for analyte molecularanions (e.g. [M−H]⁻=m/z 351.5 for LXA₄ and m/z 335.5 for LTB₄). Production mass spectra (MS/MS) were also acquired for definitiveidentification of the compounds.

Gingival CF from juvenile periodontitis patients was collected onperiostrips (Ebersole, J. L. et al. (1980) J. Periodontal Res. 15, 621).The periostrips were placed in 50 μl of phosphate buffered saline with20% Tween 20 and LXA₄, LTB₄, and PGE₂ were quantitated by specific ELISAanalyses (Neogen Corporation, Lexington, Ky.). As determined byLC/MS/MS, recoveries of known amounts of LXA₄, LTB₄, and d₄-LTB₄ fromperiostrips were linear over a 100 pg to 10 ng range, with 82.7%(r²=0.996), 85.6% (r²=0.999), and 72.7% (r²=0.996) recovery,respectively.

PMN from both healthy donors and LJP patients, incubated witharachidonic acid, produced both 5- and 15-HETE from exogenous substrate,which were identified by LC/MS/MS and chiral phase HPLC analysesindicating that the alcohol at carbon 15 position of 15-HETE was in theS configuration, suggesting involvement of a 15-lipoxygenase. On theother hand, activated PMN produced LTB₄ and its omega-oxidationmetabolite 20-OH-LTB₄, in addition to 5- and 15-HETE generated fromendogenous sources of arachidonic acid (Table 1).

TABLE 1 Eicosanoids formed by activated peripheral blood PMN* LTB₄20-OH-LTB₄ 5-HETE 15-HETE ng ng ng ng Non-periodontal 29 76 0.51 0.11disease donor LJP-001 11 42 0.00 0.46 LJP-002 28 103 0.38 0.04 LJP-00324 150 0.05 0.52 LJP-004 24 63 4.10 0.65 *PMN (5 × 10⁶) fromnon-periodontal disease donors and LJP patients were isolated andsuspended in 0.5 ml Hank's with 1.6 mM Ca²⁺ and incubated (20 min., 37°C.) in parallel with A23187 (4 μM). Samples were prepared for LC/MS/MS,and eicosanoids were identified by signature MS and MS/MS ions, and thequantifies were calculated from the recovery of the internal standard(PGB₂). Values for activated PMN from non-periodontal disease donors arerepresentative and consistent with those obtained for at least n = 6healthy donors.

Activated PMN from LJP patients, but not from controls, namelyasymptomatic individuals without evidence of clinically documentedperiodontal disease, also generated LXA₄ (FIG. 1). The presence of anumber of enzymes involved in the production of lipid mediators,including 5-, 12-, 15-LO, COX-1 and COX-2, in leukocytes fromperiodontitic patients was also confirmed by RT-PCR. CF from periodontaldisease, specifically patients with LJP were analyzed for the presenceof key eicosanoids, including LXA₄, PGE₂ and LTB₄ from each major classof eicosanoid mediators. LXA₄ was also present in the crevicular fluidof patients (Table 2), suggesting a potential role for thisimmunomodulatory molecule (Serhan, C. N. (1997) Prostaglandins 53, 107)in the local inflammatory sequelae observed within the periodontium ofpatients with periodontal diseases. Moreover, both the proinflammatoryCOX and the 5-LO derived eicosanoids, PGE₂ and LTB₄ respectively, werealso demonstrated in the CF (Tsai, C.-C. et al. (1998) J. Dentistry 26,97).

TABLE 2 Crevicular fluid from LJP patients contain eicosanoids*. PGE₂LTB₄ LXA₄ Amount: 10.2 ± 0.3 8.7 ± 0.2 1.7 ± 1.0 Ratio: 6.2 5.1 1*Values represent the mean ± SEM for 10 determinations from 4 LJPpatients (pg/μl-CF sample) from specific ELISA analyses. Ratios(relative to LXA₄) are indicated for each eicosanoid. Average CF volume:0.70 ± 0.16 μl (mean ± SEM; n = 10).

Example 2 Recruitment of Murine Leukocytes Into Air Pouches byPorphyromonas gingivalis

PMN recruitment to gingival sites and high PGE₂ levels are associatedwith periodontal disease (Offenbacher, S. et al. (1984) J. PeriodontalRes. 19, 1; Noguchi, Z., et al. (1988) Prostaglandins, Leukotrienes andEssential Fatty Acids 33, 137). We therefore sought to determine theimpact that P. gingivalis may have in vivo on leukocyte trafficking andCOX-2 expression. To this end, we used a murine air pouch model toassess leukocyte infiltration and activation.

Six to eight week old male BALB/c mice were obtained from Taconic Farms(Germantown, N.Y.). Air pouches were raised on the dorsum by s.c.injection of 3 ml of sterile air on day 0 and day 3, and all experimentswere carried out on day 6 (Sin, Y. M., et al. (1986) Ann. Rheum. Dis.45, 873). Individual air pouches (one per mouse) were injected witheither vehicle alone (0.1% ethanol), with 10 μg 15-R/S-methyl-LXA₄-me orwith 10 μg 15-epi-16phenoxy-LXA₄-me, followed by 500 μl of sterile PBSor ˜10⁵ cells of P. gingivalis strain A7436 (OD₆₀₀ 0.9-1.0) originallyobtained from the CF of a patient diagnosed with periodontitis. Micewere sacrificed 4 hr post-injection and individual air pouches werelavaged three times with sterile PBS (3 ml for each lavage) (Sin, Y. M.et al. (1986) Ann. Rheum. Dis. 45, 873; Hachicha, M.,et al. (1999) J.Exp. Med. 189, 1923). The exudates were centrifuged at 2000 rpm (5 min)and supernatants were taken and stored at −20° C. Cell pellets weresuspended in PBS (200 μl) for enumeration by light microscopy, and 50 μlof each cell suspension were mixed with 150 μl 30% BSA and thencentrifuged onto microscope slides at 500 RPM for 5 min using a cytospincentrifuge, air dried, and stained with Giemsa-Wright to identifyindividual cell type. Air pouch exudates were assessed for PGE₂ using anenzyme immunoassay (EIA) kit (Cayman Chemical Co., Ann Arbor, Mich.[cross-reactivities in the PGE₂ EIA kit were <0.04% for 6-ketoPGF_(1α and <)0.01% for LTB₄, thromboxane B₂ and arachidonic acid]). Forintravenous procedures, 100 μl of the same P. gingivalis suspension wereinjected in the orbital plexus.

In these studies, P. gingivalis elicited a massive leukocyteinfiltration into the air pouches. Approximately 10 million leukocyteswere enumerated in the P. gingivalis-injected air pouch exudates (FIG.2). This cell infiltration represents approximately three times moreleukocytes than in air pouches injected with murine TNFα (Hachicha, M.,et al. (1999) J. Exp. Med. 189, 1923). These inflammatory exudates werecomprised predominantly of neutrophilic infiltrate that represented˜80-85% of the total leukocytes recruited at 4 h. The remainder of therecruited leukocytes were mononuclear cell infiltrate ˜15-20%,consistent with earlier findings (Hachicha, M., et al. (1999) J. Exp.Med. 189, 1923), yet suggesting that P. gingivalis stimulates greaternumbers of PMN in this model than murine TNFα. Also, in the presentexperiments, relatively few leukocytes were present in PBS-injected airpouch lavages. These results showed that P. gingivalis represents apotent stimulus for the recruitment of leukocytes, predominantlyneutrophil infiltrate, within a localized site or cavity (i.e. dorsalpouch).

Example 3 Induction of COX-2 by Porphyromonas gingivalis

It is well known that COX-2 is induced in human PMN by a number ofinflammatory mediators, including Escherichia coli (Pouliot, M., et al.(1998) FASEB J. 12, 1109). We determined that P. gingivalis alsodirectly stimulates COX-2 expression in PMN freshly isolated fromperipheral blood.

Total RNA isolation and hybridization were performed essentially as inPouliot, M., et al. (1998) FASEB J. 12, 1109. Briefly, filters werehybridized with human or mouse COX-2 cDNA probes which were synthesizedby reverse-transcription polymerase chain reaction (RT-PCR). The primersused were: 5′-GCT GAC TAT GGC TAC AAA AGC TGG-3′ (SEQ ID NO. 1) and5′-ATG CTC AGG GAC TTG AGG AGG GTA-3′ (SEQ ID NO. 2) for human COX-2;5′-AAC TCC CAT GGG TGT GAA GGG A-3′ (SEQ ID NO. 3) and 5′-CCA AAG ATAGCA TCT GGA CGA G-340 (SEQ ID NO. 4) for mouse COX-2. Integrity of theRNA and equal loading on agarose/formaldehyde gels were verified byhybridization with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Theobserved COX-2 mRNA band was approximately 4.6 kb. Autoradiograms werescanned using an Epson 636 scanner (Epson America). For RT-PCR analyses,total RNA was extracted by homogenizing tissues in Trizol (Gibco-BRL,Grand Island, N.Y.), according to the manufacturer's instructions. Onepg of total RNA was used in each reaction using Titan One tube RT-PCR(Roche Molecular Biochemicals, Indianapolis, Ind.). Reversetranscription (RT) and polymerase chain reaction (PCR) were sequentiallyperformed according to the following profile: 50° C. for 30 min for RT,then 94° C. for 30 s; 60° C. for 30 s; 72° C. for 1 min; repeated 35times for PCR, followed by a final extension at 72° C. for 10 min.Primers used for mouse COX-2 were identical to those mentioned above andthe expected PCR product was 1.0 kb in length. For the detection of P.gingivalis in mouse tissues, primers specific for 16S ribosomal RNA ofthe bacteria (Genbank accession number: L16492) were utilized: 5′-GGCAGG CGG AAT TCG TGG TGT A-3′ (SEQ ID NO. 5) and 5′-GAT GTA AGG GCC GTGCTG ATT TGA-3′ (SEQ ID NO. 6). PCR products, both for P. gingivalisribosomal RNA and mouse GAPDH, had an expected length of 0.5 kb. Sampleswere migrated on 1% agarose gel containing ethidium bromide andphotographs of the gels were taken under UV illumination. Alldensitometry analyses were performed using the National Institute ofHealth Image program, which can be found at the following web site:(http://rsb.info.nih.gov) and is incorporated by reference herein.

Indeed, P. gingivalis increased the levels of COX-2 mRNA in atime-dependent fashion when compared to that from vehicle-treated PMN(FIG. 3). An increase was first observed at 1 hr and COX-2 mRNA levelsfurther increased in presence of the bacteria, compared to vehiclealone, for up to 3 hr. The expression of COX-2 in leukocytes whichmigrated into the air pouch cavity, as described in Example 2, was alsodetermined. As assessed by northern blot, COX-2 expression wasup-regulated in these activated leukocytes (see FIG. 5 insert). Theseresults indicated that oral pathogens associated with periodontaldiseases such as P. gingivalis up-regulate COX-2 expression in human PMNand in infiltrating leukocytes. These results provide direct support forthe hypothesis that neutrophils, which constitute the first and mostnumerous inflammatory cell type migrating to local gingival tissues inperiodontal disease, may constitute a previously unappreciated source ofCOX-2 derived eicosanoids, including PGE₂, in the periodontium.

Example 4 LX Analogs Inhibit Porphyromonas gingivalis-Elicited LeukocyteInfiltration and Trafficing of COX-2 In Vivo

In view of the finding that LXA₄ and aspirin-triggered LX (LX-ATL)analogs reduce leukocyte trafficking stimulated by TNFα in the murineair pouch (Hachicha, M. et al. (1999) J. Exp. Med. 189, 1923) andbecause PMN are the most abundant inflammatory cells recruited topathogen-infected gingival sites in periodontal disease, we evaluatedthe impact of the metabolically stable LX-ATL analogs 15-R/S-methyl LXA₄and 16-phenoxy LXA₄ on the recruitment of leukocytes into murine airpouches by P. gingivalis.

Air pouches (see Example 2) were injected either with vehicle, with15-R/S-methyl LXA₄ (10 μg/pouch), or with 16-phenoxy LXA₄ (10 μg/pouch)then injected with viable P. gingivalis, as in FIG. 4. In theseexperiments, LX-ATL analogs dramatically reduced the recruitment ofleukocytes into the air pouches exudate. Both analogs were essentiallyequipotent inhibitors of leukocyte infiltration, decreasing leukocyteswithin the exudates by up to 75% with as little as 10 μg application(FIG. 4).

Since PGE₂ is associated with loss of attachment and bone loss inperiodontal disease (Offenbacher, S., et al. (1992) J. Periodontal Res.27, 207), we determined PGE₂ levels in the air pouch exudates. Airpouches injected with P. gingivalis (see Example 2) contained elevatedPGE₂ levels compared to those of vehicle-injected air pouches (FIG. 5).P. gingivalis stimulated the production of nanogram levels of PGE₂ inthe exudates, which paralleled the up-regulated expression of COX-2 ininfiltrating leukocytes (FIG. 5, insert). The ATL analog 15-R/S-methylLXA₄, inhibited PGE₂ production generated in response to the oralpathogen, decreasing PGE₂ levels in the exudates by as much as 75%. Thisinhibition in PGE₂ levels paralleled the decrement in leukocytesobserved within the exudate (FIG. 4). Also, the expression of COX-2 wasevaluated in exudate leukocytes. 15-R/S-methyl LXA₄ within the air pouchdecreased the overall expression of COX-2 in exudates (FIG. 5, insert).These results indicate that P. gingivalis induces the in vivo expressionof COX-2 within infiltrating leukocytes as well as the production ofPGE₂. Moreover they indicate that LX-ATL are potent regulators of PGE₂production in air pouch exudates as a consequence of inhibitingleukocyte transmigration and reducing COX-2 mRNA levels.

Given the large number of PMN recruited at inflammatory lesions inperiodontal disease, these results identify the PMN as a potentialcellular primary target for therapeutic intervention. LX-ATL have thepotential of blocking PMN infiltration as well as reducing COX-2 derivedPGE₂ present in gingival tissues.

These findings support the concept that LXA₄, which has animmunomodulatory action, may be involved in the regulation of the localacute inflammatory responses in periodontal disease. Moreover, theseresults indicate that LX-ATL analogs, which are topically active(Takano, T. et al. (1997) J. Exp. Med. 185, 1693), are potent inhibitorsof P. gingivalis-elicited leukocyte migration towards a site ofinfection. These analogs also concomitantly reduce the overall levels ofCOX-2 mRNA associated with inflammatory exudates, which was accompaniedby a decreased production of PGE₂. Overall, these results provideevidence for a potential role for lipoxins in the host defensemechanisms evoked by P. gingivalis. Based on these findings, it can alsobe expected that lipoxin analogs would have useful for the treatment andprevention of periodontal disease.

Example 5 Systemic Up-Regulation of COX-2 Expression by Porphyomonasgingivalis.

Because COX-2 expression is up-regulated in acute and chronicinflammatory situations (Herschman, H. R. (1998) Trends Cardiovasc. Med.8, 145), we determined the systemic impact that P. gingivalis may haveby evaluating selected organ-associated levels of COX-2 mRNA. It shouldbe noted that COX-2 was initially identified as an early response gene(Herschman, H. R. et al. (1993) J. Lipid Mediat. 6, 89).

A suspension of P. gingivalis, or an equivalent volume of sterile PBS,was injected in the orbital plexus of the mice. After 4 hr, animals weresacrificed and COX-2 mRNA levels were determined in the heart and lungs.Intravenous injection of P. gingivalis caused a significant increase inthe levels of COX-2 mRNA associated with the heart and lungs (FIG. 6).P. gingivalis-specific 16S ribosomal RNA (Example 3) was readilyobserved in the heart and lungs from mice injected with the oralpathogen, and was absent in tissue samples from the PBS-injected animals(FIG. 6, insert).

These findings show that the systemic presence of P. gingivalisup-regulates expression of COX-2 (heart and lungs; FIG. 6), a marker ofon-going inflammation (Herschman, H. R. (1998) Trends Cardiovasc. Med.8, 145). The concept that a local infection by P. gingivalis may have asystemic impact on the status of the immune system was furthersubstantiated by results obtained in pilot studies, where P. gingivalisinjected in the air pouch unregulated COX-2 mRNA levels in thelung-associated tissues.

In view of these results, an effective treatment of periodontalconditions is likely to have a beneficial impact on the prognosis of anumber of systemic diseases. LXA₄ and ATL analogs reduce leukocytetrafficking stimulated by TNFα while concomitantly re-orientating thecytokine-chemokine axis towards an anti-inflammatory profile (Hachicha,M. et al. (1999) J. Exp. Med. 189, 1923). LX-ATL can thus protect hosttissues via multilevel regulation of proinflammatory signals. In view oftheir inhibitory impact on neutrophil recruitment and secondarily onPGE₂ levels, LX-ATL may be beneficial to the host not only in thecontext of periodontitis, but also in a number of diseases which involveexcessive PMN responses that can lead to losses in inflammatory barriersand increase invasion of systemic microbes.

These results demonstrate the capacity of P. gingivalis to up-regulateCOX-2 expression systemically in a murine model, supporting a potentialrole for this oral pathogen in the evolution of systemic events. Indeed,evidence is accumulating to support the notion that periodontal diseasemay affect, and worsen, systemic diseases such as coronary heartdisease, preterm labor and diabetes mellitus (Page, R. C. (1998) Ann.Periodontol. 3, 108) and that brushing or trauma to an inflamed gingivalsite can lead to septicemia (Silver, J. G., et al. (1979) J. Clin.Priodontol. 6, 33).

Therefore, the present invention, by controlling PMN responses inperiodontitis also offers a novel approach for the treatment andprevention of a variety of systemic diseases.

1. A method of treating a periodontal disease in a human or animal comprising administering to the human or animal a therapeutically effective amount of a composition comprising a compound which is a structural analog of lipoxin A₄ (LXA₄), lipoxin B₄ (LXB₄), 15-epi-lipoxin A₄ (15-epi-LXA₄) or 15-epi-lipoxin B₄ (15-epi-LXB₄).
 2. The method according to claim 1, wherein the compound is:

wherein R is selected from the group consisting of methyl and phenyl, Y is selected from the group consisting of oxygen and CH₂, and n is 1 to
 10. 3. The method according to claim 2, wherein the compound is:


4. The method according to claim 1, wherein the compound corresponds to any one of the formulae:

wherein: R is selected from the group consisting of: (i) hydrogen; and (ii) straight, branched, cyclic, saturated, or unsaturated alkyl having from 1 to 20 carbon atoms; R¹, R², R¹², and R¹³ are independently selected from the group consisting of: (i) hydrogen; (ii) straight, branched, cyclic, saturated, or unsaturated alkyl having from 1 to 20 carbon atoms; (iii) substituted alkyl having from 1 to 20 carbon atoms, wherein the alkyl is substituted with one or more substituents selected from the group consisting of halo, hydroxy, lower alkoxy, aryloxy, amino, alkylamino, dialkylamino, acylamino, arylamino, hydroxyamino, alkoxyamino, alkylthio, arylthio, carboxy, carboxamido, carboalkoxy, aryl, and heteroaryl; substituted aryl or heteroaryl, wherein the aryl or heteroaryl is substituted with one or more substituents selected from the group consisting of alkyl, cycloalkyl, alkoxy, halo, aryl, heteroaryl, carboxyl, and carboxamido; and (iv) a group Z-Y, wherein: Z is selected from the group consisting of a straight, branched, cyclic, saturated, or unsaturated alkyl having from 1 to 20 carbon atoms; substituted lower alkyl, wherein the alkyl is substituted with one or more substituents selected from the group consisting of halo, hydroxy, lower alkoxy, aryloxy, amino, alkylamino, dialkylamino, acylamino, arylamino, hydroxyamino, alkoxyamino, alkylthio, arylthio, carboxy, carboxamido, carboalkoxy, aryl, and heteroaryl; and substituted aryl or heteroaryl, wherein the aryl or heteroaryl is substituted with one or more substituents selected from the group consisting of alkyl, cycloalkyl, alkoxy, halo, aryl, heteroaryl, carboxyl, and carboxamido; and Y is selected from the group consisting of hydrogen; alkyl; cycloalkyl; carboxyl; carboxamido; aryl; heteroaryl; substituted aryl or heteroaryl, wherein the aryl or heteroaryl is substituted with one or more substituents selected from the group consisting of alkyl, cycloalkyl, alkoxy, halo, aryl, heteroaryl, carboxyl, and carboxamido; R³ selected from the group consisting of: (i) hydrogen; (ii) straight, branched, cyclic, saturated, or unsaturated alkyl having from 1 to 20 carbon atoms; (iii) substituted alkyl having from 1 to 20 carbon atoms, wherein the alkyl is substituted with one or more sub stituents selected from the group consisting of halo, hydroxy, lower alkoxy, aryloxy, amino, alkylamino, dialkylamino, acylamino, arylamino, hydroxyamino, alkoxyamino, alkylthio, arylthio, carboxy, carboxamido, carboalkoxy, aryl, and heteroaryl; substituted aryl or heteroaryl, wherein the aryl or heteroaryl is substituted with one or more substituents selected from the group consisting of alkyl, cycloalkyl, alkoxy, halo, aryl, heteroaryl, carboxyl, and carboxamido; and (iv) a group Z-Y, wherein: Z is selected from the group consisting of a branched, cyclic, or unsaturated alkyl having from 1 to 20 carbon atoms; substituted lower alkyl, wherein the alkyl is substituted with one or more substituents selected from the group consisting of halo, hydroxy, lower alkoxy, aryloxy, amino, alkylamino, dialkylamino, acylamino, arylamino, hydroxyamino, alkoxyamino, alkylthio, arylthio, carboxy, carboxamido, carboalkoxy, aryl, and heteroaryl; and substituted aryl or heteroaryl, wherein the aryl or heteroaryl is substituted with one or more substituents selected from the group consisting of alkyl, cycloalkyl, alkoxy, halo, aryl, heteroaryl, carboxyl, and carboxamido; and Y is selected from the group consisting of hydrogen; alkyl; cycloalkyl; carboxyl; carboxamido; aryl; heteroaryl; substituted aryl or heteroaryl, wherein the aryl or heteroaryl is substituted with one or more substituents selected from the group consisting of alkyl, cycloalkyl, alkoxy, halo, aryl, heteroaryl, carboxyl, and carboxamido; and R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ and R¹¹ are independently selected from a group consisting of: (i) hydrogen; (ii) halo; (iii) straight, branched, cyclic, saturated, or unsaturated alkyl having from 1 to 20 carbon atoms; and (iv) substituted alkyl having from 1 to 20 carbon atoms, wherein the alkyl is substituted with one or more sub stituents selected from the group consisting of halo, hydroxy, lower alkoxy, aryloxy, amino, alkylamino, dialkylamino, acylamino, arylamino, hydroxyamino, alkoxyamino, alkylthio, arylthio, carboxy, carboxamido, carboalkoxy, aryl, and heteroaryl; substituted aryl or heteroaryl, wherein the aryl or heteroaryl is substituted with one or more substituents selected from the group consisting of alkyl, cycloalkyl, alkoxy, halo, aryl, heteroaryl, carboxyl, and carboxamido; wherein, optionally, one or more pairs selected from R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² and R¹³ are independently replaced with a bond that forms a carbon-carbon double bond, a carbon-carbon triple bond, or a ring with the lipoxin backbone optionally substituted with one or more halo, hydroxy, lower alkoxy, aryloxy, amino, alkylamino, dialkylamino, acylamino, arylamino, hydroxyamino, alkoxyamino, alkylthio, arylthio, carboxy, carboxamido, carboalkoxy, aryl or heteroaryl groups, and wherein, optionally, one or more pairs selected from R, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R₁₂ and R¹³ are independently connected to form one or more rings containing 3 to 20 carbon atoms, wherein the rings optionally contain 1 to 6 oxygen atoms, 1 to 6 nitrogen atoms, or both 1 to 6 oxygen atoms and 1 to 6 nitrogen atoms, and wherein the rings are optionally substituted with one or more halo, hydroxy, lower alkoxy, aryloxy, amino, alkylamino, dialkylamino, acylamino, arylamino, hydroxyamino, alkoxyamino, alkylthio, arylthio, carboxy, carboxamido, carboalkoxy, aryl or heteroaryl groups.
 5. The method according to claim 4, wherein the compound is of the formula:


6. The method according to claim 5, wherein the compound is:

wherein R is selected from the group consisting of methyl and phenyl, Y is selected from the group consisting of oxygen and CH₂, and n is 1 to
 10. 7. The method according to claim 6, wherein the compound is:


8. The method according to claim 5, wherein the compound is:

wherein R is selected from the group consisting of methyl and phenyl, and Y is selected from the group consisting of oxygen and CH₂, and n is 1 to
 10. 9. The method according to claim 4, wherein the compound is of the formula


10. The method according to claim 9, wherein the compound is:

wherein R is selected from the group consisting of methyl and phenyl, and Y is selected from the group consisting of oxygen and CH₂, and n is 1 to
 10. 11. The method according to claim 10, wherein the compound is:


12. The method according to claim 4, wherein the compound is of the formula:


13. The method according to claim 12, wherein the compound is:

wherein R is selected from the group consisting of methyl and phenyl, and Y is selected from the group consisting of oxygen and CH₂.
 14. The method according to claim 1, wherein the periodontal disease is gingivitis.
 15. The method according to claim 1, wherein the periodontal disease is periodontitis.
 16. The method according to claim 1, wherein the periodontal disease is aphthous ulcer.
 17. The method according to claim 1, wherein the periodontal disease is herpetic stomatitis.
 18. The method according to claim 1, wherein the administration is topical oral administration.
 19. The method according to claim 1, wherein the composition further comprises a COX-2 inhibitor.
 20. The method according to claim 19, wherein the COX-2 inhibitor is celecoxib, rofecoxib or valdecoxib.
 21. The method according to claim 2, wherein the compound is:


22. The method according to claim 2, wherein the compound is:

wherein R is selected from the group consisting of methyl and phenyl.
 23. The method according to claim 2, wherein the compound is:


24. The method according to claim 6, wherein the compound is:


25. The method according to claim 8, wherein the compound is:


26. The method according to claim 13, wherein the compound is:

wherein R is selected from the group consisting of methyl and phenyl.
 27. The method according to claim 1, wherein the compound is 15-R/S-methyl LXA₄.
 28. The method according to claim 1, wherein the compound is 16-phenoxy LXA₄.
 29. A method of preventing a periodontal disease in a human or animal comprising administering to the human or animal a prophylactically effective amount of a composition comprising a compound which is a structural analog of lipoxin A₄ (LXA₄), lipoxin B₄ (LXB₄), 15-epi-lipoxin A₄ (15-epi-LXA₄) or 15-epi-lipoxin B₄ (1 5-epi-LXB₄). 